Angiogenesis inhibitors

ABSTRACT

A method for treating an angiogenesis-related disorder. The method includes administrating to a subject in need thereof an effective amount of a compound of the formula:  
                 
 
Each of Ar 1 , Ar 2 , and Ar 3 , independently, is phenyl, thienyl, furyl, pyrrolyl, pyridinyl, or pyrimidinyl; each of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 , independently, is R, nitro, halogen, C(O)OR, C(O)SR, C(O)NRR′, (CH 2 ) m OR, (CH 2 ) m SR, (CH 2 ) m NRR′, (CH 2 ) m CN, (CH 2 ) m C(O)OR, (CH 2 ) m CHO, (CH 2 ) m CH═NOR, or R 1  and R 2  together, R 3  and R 4  together, or R 5  and R 6  together are O(CH 2 ) m O, in which each of R and R′, independently, is H or C 1 ˜C 6  alkyl; and m is 0, 1, 2, 3, 4, 5, or 6, and n is 0, 1, 2, or 3.

RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.60/368,892, filed on Mar. 29, 2002, the content of which is incorporatedherein by reference.

BACKGROUND

Angiogenesis, formation of new blood vessels, occurs in the healthy bodyfor healing wounds and restoring blood flow to tissues after injury. Theangiogenic process is tightly controlled by various positive andnegative regulatory factors. In many disease states, the body losescontrol over angiogenesis.

Excessive blood vessel growth may be triggered by certain pathologicalconditions such as cancer, age-related macular degeneration, rheumatoidarthritis, and psoriasis. As a result of excessive angiogenesis, newblood vessels feed diseased tissues and destroy normal tissues. Incancer, the new vessels allow tumor cells to escape into the circulationand lodge in other organs.

Angiogenesis occurs via a series of sequential steps, including divisionand migration of endothelial cells that form the walls of blood vessels.About 15 proteins are known to activate endothelial cell growth andmovement. Therefore, angiogenesis can be suppressed by inhibitors ofthese activating proteins, e.g., angiogenin, epidermal growth factor,estrogen, fibroblast growth factor, interleukin 8, prostaglandins E1 andE2, tumor necrosis factor, vascular endothelial growth factor, orgranulocyte colony-stimulating factor.

Excessive angiogenesis-related disorders include cancer (both solid andhematologic tumors), cardiovascular diseases (e.g., atherosclerosis),chronic inflammation (e.g., rheutatoid arthritis or Crohn's disease),diabetes (e.g., diabetic retinopathy), psoriasis, endometriosis, andadiposity. See, e.g., Pharmacological Reviews 52: 237-268, 2001.Compounds that effectively inhibit angiogenesis are drug candidates fortreating or preventing these disorders.

SUMMARY

This invention relates to methods of inhibiting angiogenesis with fusedpyrazolyl compounds.

In one aspect, this invention features a method for treating anangiogenesis-related disorder (e.g., cardiovascular disease, chronicinflammation, diabete, psoriasis, endometriosis, or adiposity). Themethod includes administrating to a subject in need thereof an effectiveamount of a compound of the formula:

Each of Ar₁, Ar₂, and Ar₃, independently, is phenyl, thienyl, furyl,pyrrolyl, pyridinyl, or pyrimidinyl; each of R₁, R₂, R₃, R₄, R₅, and R₆,independently, is R, nitro, halogen, C(O)OR, C(O)SR, C(O)NRR′,(CH₂)_(m)OR, (CH₂)_(m)SR, (CH₂)_(m)NRR′, (CH₂)_(m)CN, (CH₂)_(m)C(O)OR,(CH₂)_(m)CHO, (CH₂)_(m)CH═NOR, or R₁ and R₂ together, R₃ and R₄together, or R₅ and R₆ together are O(CH₂)_(m)O, in which each of R andR′, independently, is H or C₁˜C₆ alkyl; and m is 0, 1, 2, 3, 4, 5, or 6,and n is 0, 1, 2, or 3. (CH₂)_(m) can be branched or linear. Note thatthe left atom shown in any substituted group described above is closestto the fused pyrazolyl ring. Also note that when there are one or more Ror (CH₂)_(m) moieties in a fused pyrazolyl compound, the R or the(CH2)_(m) moieties can be the same or different.

A subset of the above-described compounds are those in which each ofAr₁, Ar₂, and Ar₃ is phenyl or furyl. Further, each of R₁, R₂, R₅, andR₆ is H, and n is 1, e.g.,1-benzyl-3-(5′-hydroxymethyl-2′-furyl)indazole (Compound 1).

The term “Ar,” as used herein, refers to both aryl and heteroarylgroups. Aryl, e.g., phenyl, is a hydrocarbon ring system having at leastone aromatic ring. Heteroaryl is a hydrocarbon ring system having atleast one aromatic ring which contains at least one heteroatom such asO, N, or S. Examples of heteroaryl include, but are not limited to,thienyl, furyl, pyrrolyl, pyridinyl, and pyrimidinyl. Ali “Ar” maycontain one, two, three, or more substituents on its ring. In additionto those assigned to R₁, R₂, R₃, R₄, R₅, and R₆ (see above), thesubstituents can also be nitro, C₂˜C₆ alkenyl, C₂˜C₆ alkynyl, aryl,heteroaryl, cyclyl, or heterocyclyl. Alkyl, alkenyl, alkynyl, alkoxy,aryl, heteroaryl, cyclyl, and heterocyclyl, as used herein, areoptionally substituted with C₁˜C₆ alkyl, halogen, amino, hydroxyl,mercapto, cyano, or nitro. Note that the term “alkyl” refers to bothlinear alkyl and branched alkyl.

The fused pyrazolyl compounds described above include the compoundsthemselves, as well as their salts and their prodrugs, if applicable.Such salts, for example, can be formed by interaction between anegatively charged substituent (e.g., carboxylate) on a fused pyrazolylcompound and a cation. Suitable cations include, but are not limited to,sodium ion, potassium ion, magnesium ion, calcium ion, and an ammoniumcation such as teteramethylammonium ion. Likewise, a positively chargedsubstituent (e.g., amino) can form a salt with a negatively chargedcounterion. Suitable counterions include, but are not limited to,chloride, bromide, iodide, sulfate, nitrate, phosphate, or acetate.Examples of prodrugs include esters and other pharmaceuticallyacceptable derivatives, which, upon administration to a subject, arecapable of providing the fused pyrazolyl compounds described above.

The above-described compounds can also be used to treat cancer (e.g.,lung cancer). More specifically, one or more of the compounds areadministered an effective amount to a subject suffering from cancer.

As used herein, “cancer” refers to cellular tumor. Cancer cells havingthe capacity for autonomous growth, i.e., an abnormal state or conditioncharacterized by rapidly proliferating cell growth. The term is meant toinclude all types of cancerous growths or oncogenic processes,metastatic tissues or malignantly transformed cells, tissues, or organs,irrespective of histopathologic type, or stage of invasiveness. Examplesof cancers include, but are not limited to, carcinoma and sarcoma suchas leukemia, sarcomas, osteosarcoma, lymphomas, melanoma, ovariancancer, skin cancer, testicular cancer, gastric cancer, pancreaticcancer, renal cancer, breast cancer, prostate colorectal cancer, cancerof head and neck, brain cancer, esophageal cancer, bladder cancer,adrenal cortical cancer, lung cancer, bronchus cancer, endometrialcancer, nasopharyngeal cancer, cervical or hepatic cancer, or cancer ofunknown primary site.

Also within the scope of this invention are a composition containing oneor more of the fused pyrazolyl compounds described above for use intreating the afore-mentioned diseases, and the use of such a compositionfor the manufacture of a medicament for the just described treatment.

Other features, objects, and advantages of the invention will beapparent from the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the effect of Compound I on nude mice administered with aMatrigel plug containing 150 ng/mL vascular endothelial growth factor(VEGF) or basic fibroblast growth factor (bFGF).

FIG. 2 shows the effect of Compound I on nude mice implanted with A549lung tumor cells.

DETAILED DESCRIPTION

A fused pyrazolyl compound used to practice the method of this inventioncan be prepared by procedures well known to a skilled person in the art(see, e.g., U.S. Pat. No. 5,574,168). They include the followingsynthetic route: An aryl aryl ketone is first prepared by coupling anarylcarbonyl chloride with another aryl compound. Either aryl compoundis optionally mono- or multi-substituted. The ketone then reacts with anarylalkylhydrazine, the aryl group of which is also optionally mono- ormulti-substituted, to form a hydrazone containing three aryl groups. Thehydrazone group is transformed into a fused pyrazolyl core via analkylene linker, another aryl group is fused at 4-C and 5-C of thepyrazolyl core, and the third aryl group is directly connected to 3-C ofthe pyrazolyl core. Derivatives of the fused pyrazolyl compound may beobtained by modifying the substituents on any of the aryl groups.

The chemicals used in the above-described synthetic route may include,for example, solvents, reagents, catalysts, protecting group anddeprotecting group reagents. The methods described above may alsoadditionally include steps, either before or after the steps describedspecifically herein, to add or remove suitable protecting groups inorder to ultimately allow synthesis of the fused pyrazolyl compound. Inaddition, various synthetic steps may be performed in an alternatesequence or order to give the desired compounds. Synthetic chemistrytransformations and protecting group methodologies (protection anddeprotection) useful in synthesizing applicable fused pyrazolylcompounds are known in the art and include, for example, those describedin R. Larock, Comprehensive Organic Transformations, VCH Publishers(1989); T. W. Greene and P. G. M. Wuts, Protective Groups in OrganicSynthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser,Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons(1994); and L. Paquette, ed., Encyclopedia of Reagents for OrganicSynthesis, John Wiley and Sons (1995) and subsequent editions thereof.

A fused pyrazolyl compound thus synthesized can be further purified by amethod such as column chromatography, high pressure liquidchromatography, or recrystallization.

This invention features a method for treating an angiogenesis-relateddisorder (e.g., cancer or an ocular disease). The method includesadministering to a subject in need thereof an effective amount of one ormore fused pyrazolyl compounds and a pharmaceutically acceptablecarrier. The term “treating” is defined as the application oradministration of a composition including the fused pyrazolyl compoundto a subject, who has a angiogenesis- related disease, a symptom of sucha disease, or a predisposition toward such a disease, with the purposeto cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve,or affect the disease, the symptoms of the disease, or thepredisposition toward the disease. “An effective amount” is defined asthe amount of a fused pyrazolyl compound which, upon administration to asubject in need thereof, is required to confer therapeutic effect on thesubject. An effective amount of a fused pyrazolyl compound may rangefrom about 1 mg/Kg to about 100 mg/Kg. Effective doses also vary, asrecognized by those skilled in the art, depending on route ofadministration, excipient usage, and the possibility of co-usage withother agents for treating an angiogenesis-related disorder.

To practice the method of the present invention, a fused pyrazolylcompound can be administered orally, parenterally, by inhalation spray,or via an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional and intracranial injection or infusion techniques.

A composition for oral administration can be any orally acceptabledosage form including, but not limited to, tablets, capsules, emulsionsand aqueous suspensions, dispersions and solutions. Commonly usedcarriers for tablets include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added to tablets.For oral administration in a capsule form, useful diluents includelactose and dried corn starch. When aqueous suspensions or emulsions areadministered orally, the active ingredient can be suspended or dissolvedin an oily phase combined with emulsifying or suspending agents. Ifdesired, certain sweetening, flavoring, or coloring agents can be added.

A sterile injectable composition (e.g., aqueous or oleaginoussuspension) can be formulated according to techniques known in the artusing suitable dispersing or wetting agents (such as, for example, Tween80) and suspending agents. The sterile injectable preparation can alsobe a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example, as a solutionin 1,3-butanediol. Among the acceptable vehicles and solvents that canbe employed are mannitol, water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium (e.g., synthetic mono- ordiglycerides). Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions can also contain a long-chain alcohol diluent or dispersant,or carboxymethyl cellulose or similar dispersing agents.

An inhalation composition can be prepared according to techniqueswell-known in the art of pharmaceutical formulation and can be preparedas solutions in saline, employing benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

A carrier in a pharmaceutical composition must be “acceptable” in thesense of being compatible with the active ingredient of the formulation(and preferably, capable of stabilizing it) and not deleterious to thesubject to be treated. For example, solubilizing agents, such ascyclodextrins (which form specific, more soluble complexes with fusedpyrazolyl compounds), can be utilized as pharmaceutical excipients fordelivery of fused pyrazolyl compounds. Examples of other carriersinclude colloidal silicon dioxide, magnesium stearate, cellulose, sodiumlauryl sulfate, and D&C Yellow #10.

A suitable in vitro assay can be used to preliminarily evaluate theefficacy of a fused pyrazolyl compound in inhibiting the activities offibroblast growth factor (FGF) or vascular endothelial growth factor(VEGF). In vivo assays can also be performed by following procedureswell known in the art to screen for efficacious fused pyrazolylcompounds. See the specific examples below.

Without further elaboration, it is believed that the above descriptionhas adequately enabled the present invention. The following specificembodiments are, therefore, to be construed as merely illustrative, andnot limitative of the remainder of the disclosure in any way whatsoever.All of the publications, including patents, cited herein are herebyincorporated by reference in their entirety.

Synthesis of 1-benzyl-3-(5′-hydroxymethyl-2′-furyl)indazole (Compound 1)

Calcium borohydride was first prepared by stirring anhydrous calciumchloride (88.8 mg, 0.8 mmole) with sodium borohydride (60 mg, 1.6 mmole)in anhydrous THF (20 mL) for 4 hrs. Then a 30 mL THF solution containing88.0 mg 1-benzyl-3-(5′-methoxycarbonyl-2′-furyl)indazole (0.27 mmole)was added dropwise to the calcium borohydride solution at 30±2° C. Themixture was heated under reflux for 6 hrs, cooled, quenched into crushedice, placed at a reduced pressure to remove THF, and filtered to obtaina solid product. The solid was extracted with dichloromethane. Theextract was concentrated to 50 mL and a solid precipitated afterpetroleum ether was added. The precipitate was collected and purified bycolumn chromatography (silica gel-benzene) to obtain 70.0 mg1-benzyl-3-(5′-hydroxymethyl-2′-furyl)indazole at a yield of 87%.

mp: 108-109° C.

MS (%), m/z: 304 (M⁺).

IR (KBr) υ_(max): 3350 cm⁻¹ (—OH).

¹H-NMR (DMSO-d₆, 200 MHz) δ: 4.51 (2H, d, J=5.5 Hz, —CH₂O—), 5.31 (1H,t, J=5.5 Hz, —OH), 5.70 (2H, s, ═NCH₂—), 6.48 (1H, d, J=3.4 Hz, H-4′),6.97 (1H, d, J=3.4 Hz, H-3′), 7.21-7.31 (6H, m, H-5, phenyl), 7.45 (1H,t, J=8.2 Hz, H-6), 7.75 (1H, dd, J=8.2, 1.8 Hz, H-7), 8.12 (1H. dd,J=8.2. 1.0 Hz. C4-H).

Inhibition of DNA Synthesis

Human umbilical vein endothelial cells (HUVECs) were incubated in theabsence of Compound 1 (basal and control) or presence of Compound 1(with a concentration of 0.1 μM, 0.03 μM, 0.1 μM, 0.3 μM, or 1 μM).Vascular endothelial growth factor (VEGF) or basic fibroblast growthfactor (bFGF) was added (except for basal) to induce DNA synthesis,which was detected based on [³H]thymidine incorporation. The resultsshow that Compound 1 inhibited VEGF- and bFGF-induced cell proliferationof HUVECs in a concentration-dependent manner. Unexpectedly, Compound 1has IC₅₀ values of 9.0×10⁻⁸ M and 1.4×10⁻⁷ M, for VEGF and bFGF,respectively.

Additional 23 fused pyrazolyl compounds were also tested. All of theminhibited VEGF-induced cell proliferation of HUVECs, some as potent asCompound 1.

Inhibition of Tube Formation

HUVECs were cultured onto chamberslide, which was pre-coated withMatrigel (10 mg/mL). Cells were treated without Compound 1 (control) orwith Compound 1 (10 μM). VEGF (10 ng/mL) or bFGF (10 ng/mL) was added toinduce tube formation. All photos were taken at 100× magnification. Theresults show that Compound 1 inhibited VEGF- and bFGF-induced formationof networks of elongated endothelial cells.

Inhibition of Angiogenic Effect

Nude mice were subcutaneously injected with a Matrigel plug containing150 ng/mL VEGF or bFGF. Vehicle or Compound I was administrated to themice orally (1 mg/kg/day, 3 mg/kg/day, 10 mg/kg/day, 30 mg/kg/day, or100 mg/kg/day) for seven days. The angiogenic response was monitoredvisually through the transparent skin. Matrigel itself did not elicit anangiogenic response. After seven days the mice were sacrificed and theMatrigel plugs were observed in situ to quantify the ingrowth of bloodvessels. The plugs were removed, fixed in 4% formaldehyde, embedded inparaffin, sectioned at 5-μm thick for histological analysis, and bloodvessel growth quantitated by hematoxylin-eosin staining. All photos weretaken at 40× magnification. The results show that oral administration ofCompound 1 for seven days effectively inhibited VEGF or bFGF-inducedangiogenic effect in a dose-dependent manner.

In a quantitative analysis of angiogenic effect, nude mice were treatedas described above, and the plugs were removed and dissolved. Hemoglobinconcentrations were measured using a hemoglobin detection kit (SigmaChem. Co.) as indices of angiogenesis. Means ± S. E. (n=3) werepresented (see FIG. 1). Symbol “ * * * ” represents P<0.001 that arecompared with the control. The results illustrates that Compound 1effectively inhibited VEGF or bFGF-induced angiogenic effect.

Anti-Tumor Activity

10⁶ A549 lung tumor cells were introduced into the pleural space of nudemice. Compound 1 was administrated to the mice orally (10 mg/kg/day).The survival rates of Compound 1-treated mice and control mice werecompared (FIG. 2). The life span (i.e., the medium survival time) ofCompound 1-treated mice was about 1.8 times that of control mice, asanalyzed by a % T/C value [(median survival time of treatment/mediansurvival time of control)×100].

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany 1 5 combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. For example, a compound structurally analogous to a fusedpyrazolyl compound can also be used to practice the present invention.Thus, other embodiments are also within the claims.

1-15. (canceled)
 16. A method for treating cancer, comprisingadministrating to a subject in need thereof an effective amount of acompound of the formula:

wherein each of Ar₁, Ar₂, and Ar₃, independently, is phenyl, thienyl,furyl, pyrrolyl, pyridinyl, or pyrimidinyl; each of R₁, R₂, R₃, R₄, R₅,and R₆, independently, is R, nitro, halogen, C(O)OR, C(O)SR, C(O)NRR′,(CH₂)_(m)OR, (CH₂)_(m)SR, (CH₂)_(m)NRR′, (CH₂)_(m)CN, (CH₂)_(m)C(O)OR,(CH₂)_(m)CHO, (CH₂)_(m)CH═NOR, or R₁ and R₂ together, R₃ and R₄together, or R₅ and R₆ together are O(CH₂)_(m)O, in which each of R andR′, independently, is H or C₁˜C₆ alkyl; and m is 0, 1, 2, 3, 4, 5, or 6,and n is 0, 1, 2, or
 3. 17. The method of claim 16, wherein Ar₁ isphenyl.
 18. The method of claim 17, wherein Ar₂ is furyl.
 19. The methodof claim 18, wherein Ar₂ is 5′-furyl.
 20. The method of claim 19,wherein Ar₃ is phenyl.
 21. The method of claim 20, wherein each of R₁,R₂, R₅, and R₆ is H.
 22. The method of claim 21, wherein n is
 1. 23. Themethod of claim 22, wherein one of R₃ and R₄ is substituted at position2 of furyl.
 24. The method of claim 23, wherein one of R₃ and R₄ is H,and the other is CH₂OH.
 25. The method of claim 16, wherein Ar₂ isfuryl.
 26. The method of claim 25, wherein Ar₂ is 5′-furyl.
 27. Themethod of claim 26, wherein one of R₃ and R₄ is substituted at position2 of furyl.
 28. The method of claim 16, wherein Ar₃ is phenyl.
 29. Themethod of claim 28, wherein n is
 1. 30. The method of claim 16, whereinthe cancer is lung cancer.
 31. The method of claim 30, wherein Ar₁ isphenyl.
 32. The method of claim 31, wherein Ar₂ is furyl.
 33. The methodof claim 32, wherein Ar₂ is 5′-furyl.
 34. The method of claim 33,wherein Ar₃ is phenyl.
 35. The method of claim 34, wherein each of R₁,R₂, R₅, and R₆ is H.
 36. The method of claim 35, wherein n is
 1. 37. Themethod of claim 36, wherein one of R₃ and R₄ is substituted at position2 of furyl.
 38. The method of claim 37, wherein one of R₃ and R₄ is H,and the other is CH₂OH.
 39. The method of claim 30, wherein Ar₂ isfuryl.
 40. The method of claim 39, wherein Ar₂ is 5′-furyl.
 41. Themethod of claim 40, wherein one of R₃ and R₄ is substituted at position2 of furyl.
 42. The method of claim 30, wherein Ar₃ is phenyl.
 43. Themethod of claim 42, wherein n is 1.